The goal of this project is to elucidate atomic level principles by which cooperative protein function depends upon the stereochemical details of assembly. We will use three disparate hemoglobins that provide excellent systems for revealing such principles. In each case, the component subunits are folded similarly, but the extent of subunit assembly into cooperative complexes is very different. Nonetheless, there is evidence that aspects of the intersubunit interactions could be similar in all three systems. We are investigating details of the linkage between assembly and ligand binding in these three systems to learn how multiple modes of regulation are achieved within a single protein fold. We are pursuing a powerful combination of structural, functional, and mutational approaches to explore the mechanism of cooperativity in the simple Scapharca dimeric hemoglobin. A major recent finding is that ordered water molecules in the subunit interface can play a direct role in communication between subunits. High resolution crystal structures revealed that a very well ordered cluster of water molecules in the deoxy interface is disrupted upon ligand binding. Mutagenesis and osmotic experiments demonstrate that these water molecules are critical for maintenance of the low affinity conformation. This strongly suggests a mechanism for cooperativity in which the integrity of the water cluster is used as a sensor for ligation state of each subunit. We are exploring the pathway for communication that starts at the heme iron atom and passes through this water network to impact upon the second subunit. The same subunit fold found in Scapharca dimeric hemoglobin is also found in the hemoglobins of Lumbricus and lamprey, but subunit assembly is quite distinct. Nearly two-hundred subunits assemble to form Lumbricus hemoglobin, which shows both strong cooperativity and regulatory features. In contrast, lamprey hemoglobin is monomeric in the oxygenated state, but attains cooperativity from assembly into a lower affinity complex upon oxygen release and proton uptake. We are using x-ray crystallographic analysis to determine the structural basis for allostery and assembly of these molecules. The central role of cooperativity in biological function suggests that principles obtained from this study of three different, but related, allosteric proteins will have wide ranging applications to other physiologically important systems.